6 Project Summary The ability to completely sequence a single molecule of DNA by reading it directly is one of the greatest challenges of biotechnology. It also unlocks the ability to directly read RNA sequences. Many techniques have emerged in the past decade to rise to this challenge, all getting closer to this ultimate goal. Improvements are made to increase the read length, accuracy, completeness, and cost-effectiveness, but several fundamental challenges remain. Two techniques in particular are showing great progress towards this goal. The ?rst one (MinIon, Oxford Nanopore Technologies) is essentially reading the size of short sequential segments of DNA using a protein nanopore. The system is portable and features a long read length, but the error rate is large. The second technique (SMRT, PacBIO) is recording a signal generated as a polymerase complex interacts with the DNA molecule. These systems have high startup and consumable costs and a limited read length which limits the ability to analyze large-scale structural variations. The ability to accurately read an entire strand of DNA is a conditio sine qua non for complete single-molecule direct read sequencing. Reading nucleotides electronically using a pair of nanoelectrodes promises to yield single-base resolution and long read length with a minimum amount of consumables. We have developed the technology to create small graphene nanogaps that can be used as tunneling electrodes for direct-read DNA sequencing. Here, we propose to establish feasibility of a single- molecule direct read DNA sequencing platform based on graphene nanogaps. In Aim 1 we will develop a cost-effective process to create graphene nanogap tunneling electrode devices for use in direct read DNA sequencing. We will integrate our graphene devices with high frequency waveguides which enables accurate high speed performance. In Aim 2 we will develop the protocols to accurately measure long segments of single DNA molecules with single-nucleotide accuracy. We aim to increase single-nucleotide accuracy by at least a factor 10 beyond MinIon technology. We will record high frequency electronic traces of molecules passing through the nanogap and develop the signal processing pipeline to generate DNA sequences from it. We will demonstrate this system's expected unique capability by sequencing long homopolymeric sequences and sequences with long-range structural variations. A guiding principle of our design is that it should be straightforward to implement with modest invest- ment by leveraging cost-effective materials and fabrication techniques.